Journal of Chromatography B, 878 (2010) 1257–1263

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Journal of Chromatography B
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Short communication

Revised method for routine determination of urinary dialkyl phosphates using

gas chromatography–mass spectrometry夽
Jun Ueyama a,∗ , Michihiro Kamijima b , Takaaki Kondo a , Kenji Takagi a , Eiji Shibata c ,
Takaaki Hasegawa d , Shinya Wakusawa a , Tomoko Taki e , Masahiro Gotoh f , Isao Saito g
a
Program in Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan
b
Department of Occupational and Environmental Health, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
c
Department of Health and Psychosocial Medicine, Aichi Medical University School of Medicine, Aichi, Japan
d
Department of Hospital Pharmacy and Pharmacokinetics, Aichi Medical University School of Medicine, Aichi, Japan
e
COOP Aichi, Nagoya, Japan
f
Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Japan
g
Food Safety and Quality Research Center, Tokai COOP Federation, Aichi, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Among urinary organophosphorus pesticide (OP) metabolites, dialkyl phosphates (DAPs) have been
Received 4 September 2009 most often measured as a sensitive biomarker in non-occupational and occupational OP exposure risk
Accepted 8 February 2010 assessment. In our conventional method, we have employed a procedure including simple liquid–liquid
Available online 12 February 2010
extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide, PFBBr) and clean-up
(multi-layer column) for gas chromatography–mass spectrometry (GC–MS) analysis starting from 5-mL
Keywords:
urine samples. In this study, we introduce a revised analytical method for urinary DAPs; its main mod-
Organophosphorus insecticide
ification was aimed at improving the pre-derivatization dehydration procedure. The limits of detection
Urinary metabolite
Dialkylphosphate
were approximately 0.15 ␮g/L for dimethylphosphate (DMP), 0.07 ␮g/L for diethylphosphate (DEP), and
GC–MS 0.05 ␮g/L for both dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) in 2.5-mL human
Biological monitoring urine samples. Within-run precision (percent of relative standard deviation, %RSD) at the DAP levels
varying in the range of 0.5–50 ␮g/L was 6.0–19.1% for DMP, 3.6–18.3% for DEP, 8.0–25.6% for DMTP
and 9.6–27.8% for DETP. Between-run precision at 5 ␮g/L was below 15.7% for all DAPs. The revised
method proved to be feasible to routine biological monitoring not only for occupational OP exposure but
also for environmental background levels in the general population. Compared to our previous method,
the revised method underscores the importance of adding pre-derivatization anhydration for higher
sensitivity and precision.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction Environmental Protection Agency (EPA)-registered OPs are metab-

Organophosphorus compounds (OPs) have been widely and
effectively used as insecticides with applications in agricultural
settings, public health, commerce, and individual households
throughout the world [1]. Growing concern over the long-
term effects of low-level exposure to OPs on human health
has encouraged more detailed research both in experimental
and in epidemiological settings. Since about 75% of the US
夽 This paper is part of the special issue ‘Bioanalysis of Organophosphorus Toxicants
and Corresponding Antidotes’, Harald John and Horst Thiermann (Guest Editors).
∗ Corresponding author at: Department of Medical Technology, School of Health
Sciences, Nagoya University, 1-1-20 Daikominami, Higashi-ku, Nagoya, Aichi 461-
8673, Japan. Tel.: +81 52 719 1341; fax: +81 52 719 1341.
E-mail address: ueyama@met.nagoya-u.ac.jp (J. Ueyama).
olized to dialkyl phosphates (DAPs), including dimethylphosphate
(DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP),
diethylthiophosphate (DETP), dimethyldithiophosphate (DMDTP),
and diethyldithiophosphate (DEDTP) [2,3], these DAPs in urine have
been measured as biomarkers of OP exposure [4,5]. Development
of analytical equipment and protocols of sample preparation has
made it possible to detect low-level DAPs from various general
populations, revealing that OP exposure has commonly occurred
even in ordinary daily life. However, the main pathway of human
exposure (e.g. ingestion, inhalation or dermal absorption) as well as
toxicity due to long-term exposure to low-level doses still remains
to be explored. This is partly because of the difficulty in deter-
mining low-level DAPs among biological samples obtained from
the general population, due to the complicated and costly sam-
ple preparations, overall low-throughput yield, and requirement of
expensive equipment. Urine sample has been frequently used for

1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2010.02.005
1258 J. Ueyama et al. / J. Chromatogr. B 878 (2010) 1257–1263
the determination of DAP because they are collected non-invasively
and contain DAPs in higher concentrations than other biological
samples [6,7].
We previously reported a method for DAP analysis [8], which
is relatively simple and sensitive enough to be adopted in routine
biological monitoring of non-occupational as well as occupational
exposure to OPs. However, there were still some shortcomings to
be resolved such as low precision and the need for a relatively
large volume of urine (>5 mL). The aim of the present study was to
improve our previous methods [8] for urinary DAP measurement
using gas chromatography–mass spectrometry (GC–MS) equipped
with an electron ionization system and achieve higher sensitivity
and precision with high throughput and lower cost.
2. Experimental
2.1. Reagents
DMP tetramethylammonium salt (99.9% purity), DMTP ammo-
nium salt (98.9%), DEP (98.2%) and DETP ammonium salt (95.2%)
were obtained from Hayashi Pure Chemical Ind. (Osaka, Japan), and
dibutylphosphate (DBP), used for an internal standard (I.S.), was
from Tokyo Kasei Kogyo (Tokyo, Japan). Diethyl ether, acetonitrile,
n-hexane, acetone and toluene, which are pesticide residue grade,
and sodium sulfate anhydrous, sodium chloride (NaCl), sodium
disulfite (Na2 S2 O5 ) and 6 M hydrochloric acid, were purchased
from Kanto Chemicals (Tokyo, Japan). Pentafluorobenzylbromide
(PFBBr) was purchased from Sigma–Aldrich (St. Louis, MO, USA),
Florisil (60–100 mesh) from Wako Pure Chemicals (Osaka, Japan),
and Primary/Secondary Amine (PSA) 40 ␮m from Varian, Inc. (Palo
Alto, CA, USA). Water used throughout the experiments was dis-
tilled and deionized to 18 M with a Millipore Milli-Q System
(Millipore Co., Bedford, MA, USA). All other reagents were of
analytical grade purity. Muromac Mini Column (medium size,
110 mm × 8.0 mm i.d.) (Muromachi Chemical Inc., Fukuoka, Japan)
was used for dehydration and clean-up process. The clean-up col-
umn could be readily prepared without any special technique and
was composed of 0.3 g of Florisil (lower), 0.1 g of PSA (middle) and
0.5 g of sodium sulfate anhydrous (upper).
2.2. Apparatus and GC–MS conditions
Analyses of DAPs derivatized with pentafluorobenzylbromide
were performed using an Agilent 5975 inert MSD system. The GC
operating conditions were as follows: GC column, Rtx-65 (Restek,
USA), 30 m × 0.25 mm i.d., 0.25-␮m film thickness; column tem-
peratures, 70 ◦ C (1 min)–15 ◦ C/min–300 ◦ C (6 min); injection port
temperature, 250 ◦ C; carrier gas, helium (99.999% purity); flow rate,
1 mL/min. The injection volume was 1 ␮L. Splitless was changed
to split 15:1 at 2 min after sample injection. The MS operating
conditions were as follows: ionization source temperature, 230 ◦ C;
electron ionization, 70 eV; interface temperature, 300 ◦ C; injection
pressure, 88 psi. Chromatogram peak was identified by target and
quantifier ions for each pentafluorobenzyl (PFB)-DAP as shown in
Table 1. Use of C- and Q-ion presented in this table is appropriate
for selectivity and sensitivity under new analytical conditions.
2.3. Standard preparation and analytical procedure
Each standard (DMP, DEP, DMTP and DETP) was prepared
at a concentration of 1000 mg/L in methanol, and diluted with
methanol to each working standard solution. The standard solu-
tions were stored at 4 ◦ C in the dark, and were used within 1 month
of their preparation. Urine samples from healthy volunteers, who
were neither treated with any drugs nor exposed to chemicals
before collection, were used for the basic methodological exami-
nation in this study.
A flow chart of the urinary DAPs determination procedure is
shown in Fig. 1. Urine sample (2.5 mL) was pipetted into a 10-
mL screw-top glass test tube, and 20 ␮L of I.S. solution (100 mg/L
DBP), 2.5 g of NaCl, 1 mL of 6 M HCl, 50 mg of Na2 S2 O5 and 2.5 mL
of diethylether–acetonitrile (1:1, v/v) were added. After vigor-
ous mechanical shaking for 5 min, the test tube was centrifuged
(1500 × g for 5 min at room temperature). The organic phase
(upper layer) containing DAPs was passed through 1 g of anhy-
drous sodium sulfate column and collected into a new screw-top
glass test tube containing 15 mg of K2 CO3 . The aqueous phase
was re-extracted with 2.5 mL of diethylether–acetonitrile (1:1, v/v)
and then centrifuged. The supernatant obtained from the second
extraction was passed through the anhydrous sodium sulfate col-
umn, and combined with the first extract. The resulting extract was
evaporated at 40 ◦ C (heat block) to dryness with a gentle nitrogen
stream for about 1 h. To the dried extracts, 15 mg of K2 CO3 , 1 mL
of acetonitrile and 10 ␮L of PFBBr were added and incubated in a
heat block at 80 ◦ C for 30 min with occasional swirling. Afterwards,
3 mL of water and 3 mL of n-hexane were added, and the mixture
was shaken vigorously for 5 min and centrifuged for 5 min (1500 × g
for 5 min). The upper layer containing PFB-DAPs was transferred to
new test-glass tubes. The extraction was then repeated with 3 mL
of n-hexane, and the supernatant obtained from the second extrac-
tion was combined with the first extract. The combined extract was
loaded into a clean-up column, followed by washing with 5 mL of
acetone-n-hexane (2:98, v/v) for removing unreacted PFBBr. PFB-
DAPs were then eluted with 5 mL of acetone-n-hexane (15:85, v/v),
and the eluate was evaporated at 45 ◦ C to dryness with a gentle
nitrogen stream for about 15 min. The residue was dissolved in
200 ␮L of toluene and injected into GC–MS.
2.4. Assay validation
Using the proposed method, two calibration curves were sepa-
rately prepared using pooled urine. The first curve corresponded to
the concentrations of urinary DAPs ranging from 0.5 to 50 ␮g/L (four
points), and the second curve to the range from 50 to 1000 ␮g/L
(five points); the latter curve was used along with the former one
to determine the urinary DEP, DMTP and DETP in occupational OP
exposures.
To determine and calculate absolute recoveries, we spiked DAPs
at two different stages in the procedure; i.e. in the beginning of the
extraction procedure (urine sample) and prior to the derivatization
procedure. We compared the I.S. ratios obtained at these two stages.
Calibration curves were represented by the analyte/I.S. peaks
area ratio versus the concentrations of the calibration samples.
The within-run precision for our revised method was examined
through the assay of the pooled urine spiked with DAP concentra-
tions of 0.5, 2.5, 5 and 50 ␮g/L (n = 4–5). Moreover, the between-run
precision was examined through the duplicate assay of the pooled
urine spiked with DAPs at a concentration of 1, 5 and 500 ␮g/L for 4
consecutive days (n = 3–4). The limits of detection (LOD) and limit
of quantitation (LOQ) were calculated based on the signal-to-noise
ratio of 3 and 10, respectively.
2.5. Application of methods to field study samples
DAPs were measured in morning urine samples collected
from 25 healthy OP non-exposed volunteers aged 40 ± 9 years
(mean ± S.D.) in February 2009 (low-exposure urine). The meth-
ods were also applied to morning urine samples collected from 25
OP exposed persons aged 37 ± 9 years (mean ± S.D.) in August 2009
(high-exposure urine); they were all workers engaged in pest con-
trol occupation (PCO) located in the Chubu area (central Japan) and
 J. Ueyama et al. / J. Chromatogr. B 878 (2010) 1257–1263 1259

Table 1
Chemical structures, fragment ions and retention time of dialkyl phosphates and dibutyl phosphate.

Compound Structure m/za Retention time (min)

b c
C-ion Q-ion

DMP 110 306 8.86

194
DEP 258 334 9.44

197
DMTP 322 322 10.28

211
DETP 213 350 10.73

274

DBP (internal standard) 335 335 11.52

a
m/z: mass/charge ratio.
b
C-ion: selected ions for confirmation.
c
Q-ion: selected ions for quantification.

asked to provide urine samples collected on the day after insecti-
cide spraying. The reason for the difference in the sample collection
season was to verify whether the present method is feasible for a
wide range of DAP concentrations; pesticide exposure is lowest in
the general population in winter [9] and highest in PCOs in sum-
mer [10]. Collected urine samples were immediately transferred
into 10 mL polyethylene tubes and stored at −80 ◦ C until DAP assay.
The Ethics Committee of the Nagoya University Graduate School of
Medicine and Nagoya City University Graduate School of Medical
Sciences, Nagoya, Japan approved the study protocol. When uri-
nary DAP concentrations were less than LOD, they were estimated
as half the LOD value for statistical analyses [11].
3. Results and discussion
3.1. Assay validation
The validation parameters are summarized in Table 2. For
the within-run precision, percent of relative standard deviation
(%RSD) ranged from 5.6 to 27.8% for all DAPs. For the between-run
precision, the %RSD was between 7.0 and 51.3%. Reproducibility
deteriorated when the analyte/I.S. peak area ratio was not used
(data not shown). Relatively high %RSD values were shown for
the within-run precision at 0.5 ␮g/L (DMTP 25.6% and DETP 27.8%)
and between-run precision at 1 ␮g/L (DMP 36.7%, DMTP 51.3% and
DETP 23.0%). The urinary DETP concentration of 0.5 ␮g/L is near
the geometric mean in the general population [5,12]. On the other
hand, geometric means of urinary DMP and DMTP level in the
general population are around 10 ␮g/L or more [12,13]. Therefore,
biomonitoring of DETP at low concentrations is less precise in the
assessment of the OP exposure level for the general population.
Further studies are needed to accurately determine the urinary
DETP concentrations in the general population. Absolute recov-
ery data are approximately similar to those of our previous report
[8], and superior to the solid-phase extraction recovery except for
DEP [14]. The mean absolute recoveries for DMP were lower than
those for other DAPs due to its high polarity. This lower recovery
might increase the LOD value for DMP. Because absolute recover-
ies of DAPs in water differ from those in urine matrix (data not
shown), pooled urine should be used for a matrix matched cali-
bration curve. LOD and LOQ values are below or similar to those
reported by De Alwis et al. [14,15], who used solid-phase extrac-
tion and GC–MS/MS, Dulaurent et al. [16], who used liquid–liquid
extraction and LC–MS/MS, or by Ueyama et al. [8]. Concentrations
of LOQ are lower than the geometric mean of urinary DAPs in
the general populations including children and pregnant women
[12,17–19], suggesting that our present method is sensitive enough
for monitoring urinary DAPs in most general populations. The ratios
of C- and Q-ion abundances for each DAP were almost invariable at
concentrations both above and below LOD. It is unlikely that some
interference substances deteriorate selectivity for urinary DAP con-
centrations below LOD.
3.2. Advantages of this method
Advantages of the present method over the previous one were
smaller sample volume required without compromising the high
sensitivity and precision, cost-effectiveness and smaller volume of
such reagents as diethylether/acetonitrile mixture, NaCl and irri-
tating nature PFBBr.
Previously, urinary DAPs have been extracted by various
methods: liquid–liquid extraction [8,20,21], solid-phase extrac-
tion [14,15,22,23], and lyophilization [24]. Weerasekera et al. [3]
suggested that solid-phase extraction (ChemElute cartridge) and
1260 J. Ueyama et al. / J. Chromatogr. B 878 (2010) 1257–1263

Fig. 1. Analytical procedure for urinary DAPs.

analysis using the GC–MS is the most cost-effective and rapid
method. There were some drawbacks to other extraction meth-
ods; lyophilization is time-consuming and liquid–liquid extraction
is less accurate and precise [3]. If the accuracy and precision are
remediated, it should be possible to develop the liquid–liquid
extraction as a more cost-effective and highly sensitive DAP extrac-
tion procedure. We hypothesized that the derivatization procedure
using PFBBr would be adversely affected by any interference sub-
stance in extracts after the liquid–liquid extraction procedure.
It is well known that the derivatization reaction of PFBBr is
 J. Ueyama et al. / J. Chromatogr. B 878 (2010) 1257–1263 1261

Table 2
Accuracy, precision, LOD and LOQ data of analytical procedure.

Pooled urine spiked concentration (␮g/L of urine) na DMP DEP DMTP DETP

Within-run
Precision (%RSDb ) 0.5 5 19.1 18.3 25.6 27.8
2.5 4 7.6 6.3 8.0 12.3
5 4 5.6 4.1 12.5 15.1
50 4 6.0 3.6 15.6 9.6
Mean recoverye (%) 1 4 62.6 87.9 84.0 102.0
50 4 68.6 85.6 88.4 97.7
500 4 71.3 90.5 83.5 91.6

Between-run
Precision (%RSD) 1 3 36.7 16.4 51.3 23.0
5 4 15.7 10.3 9.3 7.0
500 3 10.2 15.1 9.9 11.3

R2 of calibration line
0.5–50 ␮g/L of urine 0.995 0.998 0.984 0.974
50–1000 ␮g/L of urine 0.973 0.980 0.991 0.989

LODc (␮g/L) (signal-to-noise ratio = 3) 0.15 0.07 0.05 0.05

LOQd (␮g/L) (signal-to-noise ratio = 10) 0.5 0.3 0.2 0.2
a
n: number of observations.
b
RSD: relative standard deviation.
c
LOD: limit of detection.
d
LOQ: limit of quantitation.
e
Recovery given by adding the standards on derivatization step.

inhibited in the presence of water. In the extraction procedure,
the organic phase obtained from liquid–liquid extraction using
diethylether–acetonitrile (1:1, v/v) may contain much water. The
water retained in high hydroscopic solid K2 CO3 could have affected
the efficiency of derivatizing DAPs with PFBBr, thus resulting in
the deteriorated validation data even if the post-derivatization
dry up procedure was conducted. Therefore, we examined how
the dehydration procedure prior to PFBBr derivatization affects
the accuracy, precision and sensitivity. The improvement found in
our validation data is mainly attributable to the pre-derivatization
dehydration. For example, within-run precision (RSD%) concentra-
tions at 5 ␮g/L in the present method (5.6 for DMP and 4.1 for DEP)
were lower than those in our previous method (16.1 for DMP and
20.6 for DEP) [8]. The LOD values of DAPs reported in our pre-
vious method [8] were decreased by half in the present method.
The enhanced sensitivity is likely due to improved derivatization
efficiency. While Timchalk et al. [25] adopted dehydration proce-
dure for DAP measurement in rat biological sample, the present
study is, to our knowledge, the first to indicate that the dehydration
procedure prior to derivatization can improve sensitivity, accuracy
and precision for the measurement of human urinary DAPs using
liquid–liquid extraction and PFBBr derivatization. Moreover, the

Fig. 2. Typical SIM GC–MS chromatograms in human urine samples under the LOD and near the LOQ level (A, DMP 1.0 ␮g/L; B, DEP 0.4 ␮g/L; C, DMTP 1.0 ␮g/L; D, DETP
0.2 ␮g/L), and DAP-spiked urine (E, DMP 47 ␮g/L; F, DEP 35 ␮g/L; G, DMTP 78 ␮g/L; H, DETP 22 ␮g/L). Detected masses for quantification were m/z 306 for DMP, 334 for DEP,
322 for DMTP, and 350 for DETP.
1262 J. Ueyama et al. / J. Chromatogr. B 878 (2010) 1257–1263
Table 3
Level of dialkyl phosphates in morning urine from healthy volunteers (control, n = 25) and persons engaged in pest control occupation (PCO, n = 25).
Dialkyl phosphates Detected (%) Geometric mean (␮g/L of urine) Median (␮g/L of urine)
DMP
Controls 88 4.2 8.0
PCOs 100 70.0 39.1
DEP
Controls 100 1.3 1.1
PCOs 100 7.1 5.8
DMTP
Controls 100 4.2 3.9
PCOs 100 36.0 38.5
DETP
Controls 88 0.2 0.2
PCOs 100 0.9 0.7
clean-up procedure after derivatization using a multi-layer column
effectively removed the highly irritating PFBBr and other interfer-
ing substances, resulting in a drastic reduction of unidentifiable
peaks in both the total ion chromatogram (TIC) and the selected ion
monitoring (SIM) chromatograms, and stabilization of the baseline.
Both GC–MS and LC–MS/MS methods have been developed to
detect DAPs in urine [11,15,16], and these sophisticated analyti-
cal tools have inherent advantages in terms of high selectivity and
sensitivity. The major advantage of the LC–MS/MS-based method
is its ability to analyze various metabolites simultaneously with
substantially simple sample pretreatment. However, these advan-
tages are partially counterbalanced by the higher instrument cost
in comparison with GC–MS systems. Moreover, sensitivity in DAP
measurement using LC–MS/MS [15,16] at present tends to be
lower than that using GC–MS or GC–MS/MS method [8,14,15]. In
this study we combined the liquid–liquid extraction and GC–MS
determination to achieve a lower-cost, faster and higher sample
processing capacity.
3.3. Application of methods to field study samples
Fig. 2 shows the typical chromatogram in the SIM mode of under
the LOD, near the LOQ and DAP spiked urine. Urinary concentra-
tion of DAPs in 25 controls and 25 PCOs are summarized in Table 3.
DAPs were detected in 88–100% of all human samples. Geomet-
ric mean, median, 95th percentile and maximum levels of each
DAP in the exposed group were obviously higher than those in the
non-exposed one. The urinary DAP concentrations in the exposed
workers are higher than those in our previous report [8]. The differ-
ence in the urine collection method may be the reason for this. Urine
samples were collected on the next day after insecticide spraying in
this study. But in our previous study, urine samples were collected
when the workers underwent the health checkup. The urinary DAP
concentrations in the non-exposed persons were approximately
the same or less than those in previous reports [12,13,17,18].
One disadvantage in monitoring urinary DAP is the difficulty
in estimating the OP exposure level using the measurement of
DAPs from biological samples. DAP levels determined from bio-
logical sample might be affected by the intake of environmental
DAP residue, thereby resulting in overestimation of the predicted
dose of OPs. In fact, some researchers have reported the existence
of DAPs in many foods [26]. Urinary DAP levels are usually reported
as volume-weighted concentrations (e.g., ␮g/L) or creatinine-
adjusted concentrations (e.g., ␮g/g creatinine). But the amount of
daily creatinine excretion in urine varies according to age, sex, mus-
cle mass and diet. Fortin et al. [27] suggested that the measurement
of insecticide metabolite from spot urine samples may lead to seri-
ous errors in the estimation of the actual daily absorbed doses,
even with adjustment of the creatinine contents. Further studies
95th percentile (␮g/L of urine) Maximum (␮g/L of urine)
41.3 43.7
765.9 862.0
15.8 20.1
92.3 106.8
40.7 45.9
914.9 1044.2
5.3 6.5
26.1 28.0
are needed to establish a feasible and scientifically acceptable data
collection method to reflect the 24-h total excretion level.
Our method can be applied for DAP measurement in other bio-
logical samples such as blood, hair and amniotic fluid with slight
modifications. Previously, Margariti et al. [28] determined hair
DAPs using the same technique as ours with slight modifications.
4. Conclusion
Pre-derivatization process for effective anhydration, combined
with a present GC–MS system, clearly improved sensitivity, pre-
cision, stability, and throughput without additional high-cost
requirements. The present method would allow many laborato-
ries to conduct the routine biological monitoring of urinary DAPs
in the general population, and will be helpful in epidemiological
study dealing with possible toxicity from low-level, long-term OP
exposure.
Acknowledgement
This work was supported in part by a Grant-in-Aid for Scientific
Research and a Grant-in-Aid for Young Scientists from the Japan
Society for the Promotion of Science (JSPS).
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